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At least 613 records · Page 34Linked to original sources
PubMed2003

Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator.

Genetically encoded calcium indicators, such as cameleon, have offered the promise of noninvasively monitoring activity of neurons, but no one has demonstrated whether these indicators can report calcium transients in neurons of behaving vertebrates. We show that cameleon can be expressed at high levels in sensory and spinal cord neurons in zebrafish by using neural-specific promoters in both transient expression experiments and in a stable transgenic line. Using standard confocal microscopy, calcium transients in identified motoneurons and spinal interneurons could be detected during escape behaviors produced by a touch on the head of the fish. Small movements of the restrained fish during the behavior did not represent a major problem for analyzing the calcium responses because of the ratiometric nature of cameleon. We conclude that cameleon can be used to noninvasively study the activity of neurons in an intact, behaving vertebrate. The ability to introduce an indicator genetically allows for studies of the functional roles of local interneurons that cannot easily be monitored with other approaches. Transgenic lines such as the one we generated can also be crossed into mutant lines of fish to study both structural and functional consequences of the mutations.

Animals↗
PubMed2001

Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues.

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA.

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.

Arabidopsis↗
PubMed2004

The pu.1 promoter drives myeloid gene expression in zebrafish.

PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia.

Animals↗
PubMed2003

A Link between circadian-controlled bHLH factors and the APRR1/TOC1 quintet in Arabidopsis thaliana.

APRR1 (ARABIDPSIS PSUEDO-RESPONSE REGULATOR 1) (or TOC1, TIMING OF CAB EXPRESSION 1) is believed to be a crucial component of biological clocks of Arabidopsis thaliana. Nevertheless, its molecular function remains to be fully elucidated. Based on the results of yeast two-hybrid and in vitro binding assays, we previously showed that APRR1/TOC1 interacts with certain bHLH factors (i.e. PIF3 and PIL1, which are PHYTOCHROME INTERACTING FACTOR 3 and its homolog (PIF3-LIKE 1), respectively). To critically examine the relevance of PIL1 with reference to the function of APRR1/TOC1, T-DNA insertion mutants were isolated for PIL1. No phenotype was observed for such homozygous pil1 mutants, in terms of circadian-associated events in plants. We then examined more extensively a certain set of bHLH factors, which are considerably similar to PIL1 in their structural designs. The results of extensive analyses of such bHLH factors (namely, HFR1, PIL2, PIF4, PIL5 and PIL6) in wild-type and APRR1-overexressing (APRR1-ox) transgenic lines provided us with several new insights into a link between APRR1/TOC1 and these bHLH factors. In yeast two-hybrid assays, APRR1/TOC1 showed the ability to interact with these proteins (except for HFR1), as well as PIL1 and PIF3. Among them, it was found that the expressions of PIF4 and PIL6 were regulated in a circadian-dependent manner, exhibiting free-running robust rhythms. The expressions of PIF4 and PIL6 were regulated also by light in a manner that their transcripts were rapidly accumulated upon exposure of etiolated seedlings to light. The light-induced expressions of PIF4 and PIL6 were severely impaired in APRR1-ox transgenic lines. Taken together, here we propose the novel view that these bHLH factors (PIF4 and PIL6) might play roles, in concert with APRR1/TOC1, in the integration of light-signals to control both circadian and photomorphogenic processes.

Arabidopsis↗
PubMed2004

Identification of a novel enhancer of brain expression near the apoE gene cluster by comparative genomics.

Comparative analysis of the human and mouse genomic sequences downstream of the apolipoprotein E gene (APOE) revealed a highly conserved element with previously undefined function. In reporter gene transfection studies, this element which is located approximately 42 kb distal to APOE was found to have silencer activity in a subset of cell lines examined. Analysis of transgenic mice containing a fusion construct linking this distal 631 bp conserved element to a reporter gene comprised of the human APOE gene with its proximal promoter resulted in robust brain expression of the transgenic human apoE mRNA in three independent transgenic lines, supporting the identification of a novel brain controlling region (BCR). Further studies using immunohistochemistry revealed widespread human apoE localization throughout the brains of the BCR-apoE transgenic mice with prominent expression in the cortex and diencephalon. In addition, double-label immunofluorescence performed on brain sections and cultures of primary cortical cells localized human apoE protein to cortical neurons and microglia. These studies demonstrate that comparative sequence analysis is a successful strategy to predict candidate regulatory regions in vivo, although they do not imply that this element controls apoE expression physiologically.

Animals↗
PubMed2007

Multiple upstream modules regulate zebrafish myf5 expression.

BACKGROUND: Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. RESULTS: We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP) reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1) the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2) the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3) the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4) the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5) the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. CONCLUSION: We suggest that the cell lineage-specific expression of myf5 is delicately orchestrated by multiple modules within the distal upstream region. This study provides an insight to understand the molecular control of myf5 and myogenesis in the zebrafish.

Animals↗
PubMed2001

Evidence for expression level-dependent modulation of carbohydrate status and viral resistance by the potato leafroll virus movement protein in transgenic tobacco plants.

High-level constitutive expression of the cell-to-cell movement protein from the phloem-restricted potato leafroll virus (PLRV-MP17) in transgenic tobacco plants leads to growth retardation and severe phenotypic changes of source leaves paralleled by a drastic accumulation of soluble sugars and starch (Herbers et al., 1997). To investigate whether the MP17-induced alteration in carbon metabolism is related to the targeting and modification of specific plasmodesmata (Pd) or is rather due to pleiotropic effects caused by high MP17 protein amounts, non-phenotypic tobacco plants expressing a MP17:GFP fusion protein were obtained and compared with previously described MP17 transgenic lines. Confocal laser scanning microscopy and immunogold labelling studies revealed an overall affinity of MP17 to Pd in vascular and non-vascular tissue of source leaves, whereas in sink leaves GFP fluorescence was restricted to Pd of trichomes. In source leaves, plasmodesmal size exclusion limits of mesophyll cells were likewise increased by MP17 and MP17:GFP independent from steady-state levels of the protein amount and phenotypic alteration. Conversely, carbohydrate contents in source leaves strictly correlated with quantified MP17 protein levels. Low expression of MP17 and MP17:GFP decreased soluble sugars and starch contents in leaves possibly due to changes in plasmodesmal permeability while increasing MP17 protein levels led to carbohydrate accumulation and a stunted growth. Infection of transgenic lines with the unrelated potato virus Y (PVY)N revealed an expression level-dependent mode of MP17-mediated resistance. Phenotypic changes and carbohydrate-mediated defence responses as indicated by elevated levels of PR-protein transcripts were crucial for increased viral resistance, whereas plasmodesmal targeting and modification by MP17 per se had either no effect or even increased susceptibility to PVY. Thus, our results implicate that the absolute level of expression needs to be critically considered when elucidating the effect of MPs on carbon metabolism, biomass allocation and virus resistance.

Base Sequence↗
PubMed2005

Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance.

To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.

Acclimatization↗
PubMed1997

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants.

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.

Avena↗
PubMed2002

Generating new marine cell lines and transgenic species--conference summary.

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.

Animals↗
PubMed2007

Identification of a unique transgenic mouse line that develops megabladder, obstructive uropathy, and renal dysfunction.

Urinary tract malformations, obstructive uropathy, and hypoplasia/dysplasia are extremely important in terms of pediatric health care costs, with end-stage renal failure in children estimated to cost >$15 billion annually in the United States alone. Even so, little is known regarding the mechanisms that control these processes. Identified was a unique mutant mouse model that develops in utero megabladder, resulting in variable hydroureteronephrosis and chronic renal failure secondary to obstructive uropathy. These animals, designated mgb for megabladder, possess a primary defect in bladder smooth muscle development that is apparent by embryonic day 15. The mgb mouse represents an excellent model for the study of normal and pathogenic bladder development, including the postnatal progression of chronic renal failure that results from the development of in utero obstructive uropathy.

Animals↗
PubMed1999

Peripheral axotomy slows motoneuron degeneration in a transgenic mouse line expressing mutant SOD1 G93A.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that involves motoneuron degeneration, paralysis, and death. Mutations in Cu, Zn superoxide dismutase (SOD1) are one cause of this disease. It has been a puzzle as to why mutations in SOD1, an enzyme expressed in many neuronal types, selectively kill motoneurons. To begin to explore the factors that determine this selectivity, we carried out peripheral axotomy in mice expressing a mutant SOD1 (G93A mice) and controls (nontransgenic mice and mice expressing wild-type human SOD1). Axotomy in controls induced a predicted axonal atrophy and a small degree of axon loss. The axonal atrophy led to an increase in the number of small axons and a decrease in the number of large axons. In contrast to the controls, axotomy in G93A mice reduced the extent of axon degeneration at the end stage of the disease, leading to an increase in the number of surviving motor axons. Interestingly, all of the increased surviving axons were in the axon group with diameters smaller than 4.5 microm. This result suggests an apparent threshold of vulnerability that is correlated with axon size.

Amino Acid Substitution↗
PubMed2006

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterless gus::nptII fusion gene based vector.

We have generated putative promoter tagged transgenic lines in Arachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated by Agrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4mg/l in combination with 0.1 mg/l alpha -napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age, Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated with Agrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in beta-glucuronidase (GUS) assays. Among the in vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T 0 plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T 0 plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T 0 and corresponding T 1 progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.

Arachis↗
PubMed1996

A promoter identified in the 3' end of the Ac transposon can be activated by cis-acting elements in transgenic Arabidopsis lines.

In experiments directed to develop a promoter trap strategy in Arabidopsis, using a Ds chimaeric element containing a promoterless beta-glucuronidase (GUS) gene, we identified a promoter in the 3' end region of the Ac transposable element. The promoter initiates most of the transcripts at coordinate 4250 in the Ac sequence and is oriented towards the internal part of the element. When fused to a promoterless GUS gene, the promoter allows transient expression in Arabidopsis leaves. After stable integration into the Arabidpsis genome, no GUS activity was observed in most of the transformed lines analysed. Only two of them exhibited different tissue-specific GUS expression. When a CaMV 35S promoter was introduced into the transformation vector, downstream to the reporter gene, a high level of GUS activity was observed in all the transformants. These results strongly suggest that the promoter is not normally expressed at a significant level in Arabidopsis transformed lines except when activated by neighbouring cis-acting enhancer elements. This opens an interesting possibility for using this promoter to develop 'enhancer trap' strategies in Arabidopsis. Since only one Ac transcript, initiating in the 5' end region of the element has been reported to date in maize, the putative biological function of the promoter remains an open question.

Animals↗
PubMed1993

A variety of epistatic interactions can occur between partially homologous transgene loci brought together by sexual crossing.

Epistatic interactions between unlinked transgene loci in tobacco plants were studied following sexual crosses between different transgenic lines. Three potential "modifier" transgene loci, which were structurally similar but integrated at different chromosomal locations, were tested for their ability to influence the expression of a partially homologous "target" transgene locus. After introduction of an individual modifier locus, the target locus could be either unaffected, completely inactivated and methylated or differentially sensitive, showing more complete inactivation and methylation when homozygous than when hemizygous. The implications of these results for inbreeding depression in plants are discussed.

Cinnamates↗
PubMed1994

Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging.

We have introduced a genetically marked Dissociation transposable element (DsHPT) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed DsHPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying DsHPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using DsHPT, we examined the genomic distribution of DsHPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers beta-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed DsHPT elements. RFLP mapping of 21 transposed DsHPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.

Agrobacterium tumefaciens↗
PubMed2006

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

Acyltransferases↗